Innovative particle and column technology for the high ......TOSOH BIOSCIENCE LLC Supelco Seminar Series, 2011 Canada Innovative particle and column technology for the high throughput

TOSOH BIOSCIENCE LLC Supelco Seminar Series, 2011 CanadaTOSOH BIOSCIENCE LLC

Innovative particle and column technology for the high throughput

LC-MS analysis of biomolecules

Atis Chakrabarti, Ph.D.Technical Service Specialist

TOSOH BIOSCIENCE LLC Supelco Seminar Series, 2011 Canada 2

• Size (Molecular Weight)• Molecular Charge

• Hydrophobic Regions

• Conformational Recognition Sites

• Size Exclusion• Anion Exchange

Cation ExchangeIon Pair

• Normal PhaseReversed PhaseHydrophobic Interaction

• AffinityChiral

Sample Characteristics and Mode of Chromatography


Liquid Chromatography and Mass Spectrometry

• Liquid chromatography

Fundamental separation technique in bioscience, chemistry and related field Suitable for nonvolatile compounds Suitable for compounds prone to breakdown at high

temperatureWidely used for small molecules and macromolecules

• Mass spectrometry

Signal strength Mass


Advantages of Mass Spectrometry

• High sensitivity

• High specificity

• Doesn’t need a chromophore in the analytes

• Able to analyze poorly resolved peaks also

Liquid chromatography coupled with mass spectrometry, is a very useful analytical tool not only for identification but also for quality control purpose in qualitative and quantitative analyses.

Mass spectrometry is used in forensic toxicological analysis.

TOSOH BIOSCIENCE LLC Supelco Seminar Series, 2011 Canada

Factors in analysis – a puzzle

5


Concerns for LC-MS

● Concentration of the sample

● Mobile phase compatibility select the solvents and buffers carefully.

● Interference coming from mobile phase or sample preparation with regards to ionization and signal strength need to be optimized

● The column


Advantages of small particle size columns

●Higher efficiencyShorter diffusion path length through the particles

●Increased resolution

●Optimization of the extra-column components can significantly improve column performance.Reduce ID capillary tubing, detector cell volume Select a fast detector response timeReduce sample injection volume (< 2mm ID)

7


SEM of HPLC Column Packing materials

8


Advantages of small particle size columns

●Smaller particles are more efficient

at the expense of higher back pressure compared to columns packed with larger particles.

usually traded off by reducing column lengtho shorter analysis timeso lower column back pressure

●Three micron packed columns are now a common tool in many LC-MS applications.

9


• Polar stationary phase similar to normal phase

• Mobile phase similar to reversed phase (high % organic)

• Elution in order of increasing hydrophilicity

Bonded phase of HILIC Column

TSKgel Amide-80TSKgel NH2-100


Bonded phase structure in Ion Exchange chromatography

TSKgel STAT columns

•Very efficient chromatography•for high as well as low MW solutes

•novel bonding chemistry and the absence of micro-pores• High speed and high resolution analysis of biomolecules• Higher adsorption capacities and lower pressures compared with competitive non-porous columns• 7 or 10µm particles for SP and CM chemistries


Three micron packed TSKgel ODS-100V and ODS-100Z columns are effective tools to increase throughput and improve precision in LC-MS applications.

Bonded phases in Reversed phase Chromatography


Bonded phases in Hydrophobic Interaction Chromatography (HIC)

13


Gel Filtration Chromatography (GFC) Column Selection

Tota

l exc

lusi

on

Tota

l Inc

lusi

on

Pore volume



Molecular mass separation ranges (Da) of TSKgel SWXL Series

Column Polyethylene glycol(linear)

Dextran(branched)

Protein(globular)

G2000SWXL 500~15,000 1,000~30,000 5,000~100,000

G3000SWXL 1,000~35,000 2,000~70,000 10,000~500,000

G4000SWXL 2,000~250,000 4,000~500,000 20,000~7,000,000


Proteins (general)Select appropriate pore size based on knowledge or estimate of

protein MW

Protein of unknown molecular weightTSKgel G3000SWXL

Ideal investigational column (Scouting column)

If peak elutes near the exclusion volumeSwitch to TSKgel G4000SWXL

If peak elutes near the end of the chromatogramSwitch to TSKgel G2000SWXL



Analysis of purified mAb

Mobile phase: 0.1mol/L Phosphate buffer (mono/dibasic), 0.1mol/L NaCl, 0.05% NaN3, pH 6.8; Flow rate: 1mL/min; Detection: UV@280nm (micro flow cell); Injection volume: 5µL

7.8mm ID x 30cm 5µm, F=1mL/min

4.6mm ID x 30cm 4µm, FR 0.35mL/min

Both runs are at same linear velocity


Dependence of HETP on sample loadSample: Bovine Serum Albumin (BSA)

~250µg~250µg


The Characterization of Biological Samples by Microbore TSKgel SuperSW3000 SEC Columns

Fraction of interest analyzed by off-line SELDI/TOF/MS to establish presence of BSA aggregates and IgG

Column: TSKgel SuperSW3000 microbore, 1mm ID

• excellent choice for the rapid separation of proteins and enzymes at micro scale

• a great fit for the trace analysis of biological components by LC-MS.


LC/MS Chromatogram (TIC) of -lactoglobulin Tryptic Digest

In this analysis we have used a highly consistent and chemically stable silica-based octadecylsilane stationary phase (TSKgel Super-ODS column) for LC-MS


TSKgel Super Series Columns

• Increased efficiency

• Reduction in analysis time

• Higher resolution

• TSKgel Super Series columns are available in: 1, 2, or 4.6mm ID 5 or 10cm length

• These ultra efficient columns are coupled with the specificity of Mass Spec, which results in superior analytical power.


Three micron packed TSKgel ODS-100V and ODS-100Z columnsare effective tools to increase throughput and improve precision inLC-MS applications.

TSKgel ODS-100V and 100Z Columns


Rapid Identification of 20 Peptides

• Unwanted secondary ionic interactions from residual silanols can be eliminated by adding trifluoroacetic acid (TFA) to the mobile phase.

• The use of “mass-spec friendly TFA” eliminates extra steps involved with removing salts or non-volatile acids required by amino-bonded columns to eliminate ionic interactions.


TIC on TSKgel Amide-80, 3µm Column with LC/ESI/MS under Low pH Mobile Phase Conditions

Low level of background noise (indication of low bleeding) was observed in the total ion chromatogram (TIC) for LC/ESI/MS using an acidic mobile phase containing 0.1% formic acid with a gradient elution method. No sample was injected.

This data suggests that TSKgel ODS-100V, 3µm columns are well suited for LC-MS applications.


• Polar stationary phase similar to normal phase

• Mobile phase similar to reversed phase (high organic)

• Elution in order of increasing hydrophylicity

Bonded Phase HILIC Columns

TSKgel Amide-80TSKgel NH2-100


Both can be used for with evaporative light scattering (ELS) and mass spec (MS) detectors.

The 3µm material – for use in LC/MS applications for the analysis of active pharmaceutical ingredients and their metabolites.

TSKgel NH2-100: A Stable Amino Column


Columns: TSKgel NH2-100, 3µm ,4.6mm ID x 15cmTSKgel Amide-80, 3µm, 4.6mm ID x 15cm

Eluent: H2O/ACN=10/90~90/10 (vol.%)Flow rate: 1.0mL/minTemp: 40ºC Detector: RISample: inositol Injection vol.: 10µL

TSKgel NH2-100 - Expanded HILIC Selectivity


Identification of Isobaric Glycoforms by Retention Time (Glycobase) and MS/MS Experiments

Protein construct of the zp domain of murine tgfr-3 expressed in HEK293EBNA


Separations of 2-AB Labeled N-glycans

The structure analysis was completed by high resolution mass spectra acquired on a MALDI QIT TOF MS instrument.

Fluorescence chromatograms of HILIC separations of 2-AB labeled N-glycans released from the recombinant ZP domain construct of murine TGFR-3, were compared to the dextran ladder.


Separations of 2-AB Labeled N-glycans


MALDI Mass Spectrum of 2-AB-labeled Glycans Released from ZP domain Construct of Murine TGFR3


MS2 (CID) Mass Spectrum of m/z 2243


MS3 (CID) Mass Spectrum of m/z 1930


-5.0E+03

0.0E+00

5.0E+03

1.0E+04

1.5E+04

2.0E+04

0 5 10 15 20Retention time, min

Inte

nsi

ty, c

ps

His 77.4µg/L

1.6µg/L

-1.0E+02

0.0E+00

1.0E+02

2.0E+02

3.0E+02

4.0E+02


Inte

nsi

ty, c

ps

Spd

0.6µg/L

Biogenic Amines in Tuna as Function of Storage

-1.0E+03

0.0E+00

1.0E+03

2.0E+03

3.0E+03

4.0E+03

5.0E+03


Inte

nsi

ty, c

ps

Put

3.6µg/L

-2.0E+03

0.0E+00

2.0E+03

4.0E+03

6.0E+03

8.0E+03

1.0E+04


Inte

nsi

ty, c

ps

Tyr 5.0µg/L

0.1µg/L

-2.0E+02

0.0E+00

2.0E+02

4.0E+02

6.0E+02


Inte

nsi

ty, c

ps

Cad 11.7µg/L

-1.0E+02

0.0E+00

1.0E+02

2.0E+02

3.0E+02


Inte

nsi

ty, c

ps

Trp 0.2µg/L

0.2µg/L

Stored at room temperature for 2 daysStored frozen


TSKgel STAT Ion Exchange Columns

•Very efficient chromatography•for high as well as low MW solutes

•novel bonding chemistry and the absence of micro-pores• High speed and high resolution analysis of biomolecules• Higher adsorption capacities and lower pressures compared with competitive non-porous columns• 7 or 10µm particles for SP and CM chemistries


Basic Properties of TSKgel STAT Columns

TSKgel Q-STAT TSKgel SP-STAT TSKgel CM-STAT TSKgel DNA-STATHydrophilic Hydrophilic Hydrophilic Hydrophilic

non-porous resin non-porous resin non-porous resin non-porous resin7, 10µm 7, 10µm 7, 10µm 5µm

(mono-disperse) (mono-disperse) (mono-disperse) (mono-disperse)Ligand Quaternary Sulfopropyl Carboxymethyl Quaternary

3.0 x 3.5 3.0 x 3.5 3.0 x 3.54.6 x 10 4.6 x 10 4.6 x 10

Base materials

Particle size

Column dimensionsmm ID x cm 4.6 x 10


Protein Separations on Non-Porous Anion Exchange Columns

Improved protein peak shapes on TSKgel Q-STAT vs. non-porous WAX column.

Commercial WAX column4.0mmI.D. x 25cm

Rs = 10.1

TSKgel Q-STAT 4.6mmI.D. x 10cm (7um)

Rs = 15.3


Proteins on Non-Porous Cation Exchange Columns

In this comparison of protein separations on various cation exchange columns, different selectivities were observed for each set of proteins on all three columns. The TSKgel SP-STAT column shows excellent resolution for cytochrome C and lysozyme.

Commercial WCX column4.0mmI.D. x 25cm

TSKgel CM-STAT 4.6mmI.D. x 10cm (7um)

TSKgel SP-STAT 4.6mmI.D. x 10cm (7um)

1

1

1

2

2

2

3

3

3

4

4

4

5

5

5


Fast Protein Separations on Monolithic and Non-Porous Cation Exchange Columns

Commercial monolithic5.0mmI.D. x 5cm

TSKgel SP-STAT 3.0mmI.D. x 3.5cm (10um)

1 2 3

1 2 3


Separation on Toyopearl GigaCap resins

40


* Toyopearl GigaCap is a registered trademark of Tosoh Corporation

Toyopearl GigaCap S-650M is the first in a family of ion exchange resins optimized for high throughput chromatography of IgG.

The high capacity for lysozyme shows that Toyopearl GigaCap S-650M will be an excellent resin for smaller proteins as well.


The new Toyopearl GigaCap

S-650M resin featured very high

binding capacities with excellent

binding kinetics and almost

quantitative recoveries (data not

shown).

75 >200


Conclusions

• The new surface modifications improve not only

chromatographic performance but also are effective

tools to increase throughput and improve precision

in LC-MS applications.

TOSOH BIOSCIENCE LLC Supelco Seminar Series, 2011 CanadaTOSOH BIOSCIENCE LLC

Innovative particle and column technology for the high throughput

LC-MS analysis of biomolecules

Atis Chakrabarti, Ph.D.Technical Service Specialist

Documents

Innovative particle and column technology for the high ......TOSOH BIOSCIENCE LLC Supelco Seminar Series, 2011 Canada Innovative particle and column technology for the high throughput